Modulation of Cytoskeletal Associated Proteins Regulates Cell Death and Survival in Uveal Melanoma

Abstract

Purpose: Purpose: Approximately 50% of Uveal Melanoma (UM) patients will be diagnosed with liver metastases within 5-years of diagnosis. Micro-metastases are present in the liver as early as 2-years before diagnosis of the intraocular malignancy. Recent work from our laboratory suggests that inhibiting the upregulation of cytoskeletal signaling could offer potential therapeutic targets for metastasis control. In this study we elucidate the mechanism(s) involved.

Methods: Methods: We performed gene transcription, flow cytometry as well as cellular and molecular biology analyses after pharmacological modulation of paxillin Y118 using the paxillin inhibitor 6-B345TTQ. The effects of paxillin inhibition (pY118) were measured on pAKT, Protein Kinase C-delta (PKC-δ), pro-apoptotic molecules expression, actin polymerization and cellular proliferation in UM cell lines and immunohistochemical (IHC) analyses in normal and with UM. We performed cutting edge Nano3D (Nano 3D Biosciences Inc, Houston, TX) technology to investigate changes in cellular migration and proliferation after paxillin inhibition.

Results: Results: We observed an increase in the alpha-4 subunit of VLA-4 and paxillin both in the UM cell lines by flow cytometry analysis (p<0.05) and in UM eyes compared to normal eyes by IHC. Targeting of paxillin through inhibition of pY118 reduced cellular proliferation in primary, not metastatic, UM. Mechanistically, we observed a significant reduction in pAKT signaling in metastatic UM (p<0.05) by Western blot analysis and phospho-PKC-δ in the metastatic UM cell lines by flow cytometry. IHC analysis in human eyes revealed differences in PKC-δ expression in normal and UM eyes.

Conclusions: Conclusions: Our in vitro results identified paxillin as a potential therapeutic target to arrest UM cellular proliferation and migration. Inhibition of paxillin preferentially affected primary UM cells compared to metastatic cell lines through pAKT and PKC-δ and may represent a potential targeted therapy for micrometastases.